#Immediately after preperation cells were tested wtihout being frozen in liq N2 with Puc19, P1010 in pSB1A2, and a Neg Control. 270uL were plated

+

*The Neg control grew nothing

+

*Puc19 grew 30 colonies (low yield due to <.5 uL used to transform

+

*P1010 in pSB1A2 created many.

+

#PCR was done on BBa_I741015 and BBa_171017 using both Taq and PFx Polymerase

+

*Results were:

+

Taq=success

+

Pfx=no DNA found (except primers)

+

+

<h2>'''June 28, 2007: PCR on Promoter Regions'''</h2>

+

'''PCR Setup:'''

+

#10X Pfx Amplification Buffer 5 μl/10x Thermo Polymerase Buffer 5uL

+

#10 mM dNTP mixture* 1.5 μl

+

#50 mM MgSO4 1 μl

+

#Primer mix (10 μM each)* 1.5 μl

+

#Chromosomal DNA 2ul

+

#Platinum® Pfx DNA Polymerase 0.5 μl/TAq Polymerase .5uL

+

#Autoclaved, distilled water 38.5 μl

+

+

Reaction:

+

#+Pfx Primers 3398+3399 '''Part:BBa_I741015'''

+

#+Pfx Primers 3400+3401 '''Part:BBa_I741017'''

+

#+Pfx Primers 3406+3407 '''Part:BBa_I741018'''

+

#+Pfx Primers 3408+3409 '''Part:BBa_I741019'''

+

#+Pfx Primers 3402+3403 '''Part:BBa_I741020'''

+

#+Pfx Primers 3404+3405 '''Part:BBa_I741021'''

+

#+TAq Primers 3398+3399 ''Part:BBa_I741015''

+

#+TAq Primers 3400+3401 ''Part:BBa_I741017''

+

#+TAq Primers 3406+3407 ''Part:BBa_I741018''

+

#+TAq Primers 3408+3409 ''Part:BBa_I741019''

+

#+TAq Primers 3402+3403 ''Part:BBa_I741020''

+

#+TAq Primers 3404+3405 ''Part:BBa_I741021''

+

#-TAq Primers 3398+3399 ''Negative Control''

+

#-PFx Primers 3398+3399 ''Negative Control''

+

+

<h2>'''June 29, 2007: PCR using Pfx Thermal Gradient Test'''</h2>

+

'''PCR Setup:'''

+

#10X Pfx Amplification Buffer 5 μl/10x Thermo Polymerase Buffer 50uL

+

#10 mM dNTP mixture* 15 μl

+

#50 mM MgSO4 10 μl

+

#Primer mix (10 μM each)* 15 μl

+

#Chromosomal DNA 20ul

+

#Platinum® Pfx DNA Polymerase 6 μl/TAq Polymerase .6uL

+

#Autoclaved, distilled water 38.5 μl

+

+

Reaction:

+

#+Pfx Primers 3398+3399 '''Part:BBa_I741015'''

+

#+Pfx Primers 3400+3401 '''Part:BBa_I741017'''

+

#+Pfx Primers 3406+3407 '''Part:BBa_I741018'''

+

#+Pfx Primers 3408+3409 '''Part:BBa_I741019'''

+

#+Pfx Primers 3402+3403 '''Part:BBa_I741020'''

+

#+Pfx Primers 3404+3405 '''Part:BBa_I741021'''

+

#+TAq Primers 3398+3399 ''Part:BBa_I741015''

+

#+TAq Primers 3400+3401 ''Part:BBa_I741017''

+

#+TAq Primers 3406+3407 ''Part:BBa_I741018''

+

#+TAq Primers 3408+3409 ''Part:BBa_I741019''

+

#+TAq Primers 3402+3403 ''Part:BBa_I741020''

+

#+TAq Primers 3404+3405 ''Part:BBa_I741021''

+

#-TAq Primers 3398+3399 ''Negative Control''

+

#-PFx Primers 3398+3399 ''Negative Control''

+

+

<h2>'''July 5, 2007: Plan and Motility Preperations'''</h2>

+

'''plan'''

+

#Motility:

+

##Miniprep Sender and Receiver cells (elute with 30uL)

+

##*Digest with ECO and SPE

+

##*Run Gel/Extract

+

##*Ligate Sender with psB1A2/transform

+

##*Digest with Xba and PST

+

##*Run Gel/Extract

+

##*Ligate sender with psB1A2

+

##Make 16x Eiken Agar Plates

+

##Transform low copy plasmid (amp)

+

##*Prepare to add Receiver into the amp plasmid

+

#Diauxie:

+

##Plate rest of transformation

+

##Retry Ligation and transformation

+

##Redigest pSB1A2

+

##*Gel/extract

+

##*Ligate with extracted I741015

+

##Attempt PCR with Pfx on 4 different temperatures

+

##Redigest I741015/pSB1A2 with

+

##*1uL SPE

+

##*.5uL XBA

+

##*For 20 hours

+

#Misc:

+

##pH new water and autoclave

+

##Prepare more LB amp plates

+

+

'''Motility Information'''

+

+

<h2>'''July 6, 2007: Motility Assay'''</h2>

+

'''Experiment Aims'''

+

+

Test motility at a variety of different temperatures.

+

+

'''Procedure'''

+

+

Eiken Agar Plates were made using a modified version of general procedure [[IGEM:PennState/2006/Eikenplates|1]]. An ncreased agar concentration was made to increase durability of plate and reduce the motility.

+

+

''.28% Eiken Agar Plates:''

+

*.25g Bactotryptone

+

*.20g NaCl

+

*.15g sodium citrate

+

*.07g Eiken Agar (for 20EikenAgar)

+

+

#Receiver cells grown at 37 degrees to OD500 ~1.2

+

#*Rediluted to OD500 ~ .2 and grown for 1.5 hours at 30 degrees

+

#.3uL plated of receiver cells with 1uL of sender cells (.3uL used with wildtype RP4037)

Eiken Agar Plates were made using a modified version of general procedure [[IGEM:PennState/2006/Eikenplates|1]]. An ncreased agar concentration was made to increase durability of plate and reduce the motility.

+

+

''.24-.32% Eiken Agar Plates:''

+

*.25g Bactotryptone

+

*.20g NaCl

+

*.15g sodium citrate

+

*.varied amount of Eiken Agar

+

+

+

Controls

+

*Motile Strain RP4037

+

*+HSL Plate

+

*-Sender Cell Plate

+

+

'''Results:'''

+

+

insert pictures here

+

+

'''Observations:'''

+

+

.24% plates seem too fragile to use.

+

.26-.32% seem fine

+

+

'''Conclusions:'''

+

+

use a .30% Eiken Agar Plate

+

+

<h2>'''July 10, 2007: Changing the sender to a high copy plasmid'''</h2>

+

Part BBa_F1610 was grown up off registry plate and transformed into DH5a.

+

Motility assay will be retried Wednesday.

+

+

Digested P1010 PSB1A2

+

Gel extracted

+

Ligated with

+

Extracted products 7/10 in LW-1

+

Transformed

+

+

+

<h2>'''July 16, 2007: Testing what we have and preparing for sequencing'''</h2>

+

Grew up 6-7 colonies per plate of ligations. (large numbers on negative plates, thus a quck miniprep, and subsequent digestion should reveal if the constructs were made.

+

+

Also, a gigantic gel was made to test the dna concentration of everything we have. The full list can be found in Noah's lab notebook.

+

+

<h2>'''July 17, 2007: Giant Digest'''</h2>

+

+

+

32 +4X

+

0.25u EcoRI

+

0.25u SpeI

+

1.00u EcoRI Buffer (10x)

+

3.00u DNA Miniprep

+

5.50u H2O

+

+

Mix:

+

9uL EcoRI

+

9uL SpeI

+

36uL EcoRI Buffer

+

DNA to be added

+

198uL H2O

+

+

<h2>'''July 23, 2007: Motility Assay Protocol'''</h2>

+

Motility Protocol:

+

# For each plate to be made, add the following to a 125mL flask:

+

*0.25g Bacto tryptone

+

*0.20g NaCl

+

*0.15g sodium citrate

+

*0.075g Eiken agar (for a 0.30% agar swarm plate)

+

note: <.25% plates are difficult to deal with as they break very easily (Weiss motility assay 3/11/07) .26%-.32% is what I tend to use. Cells did not swarmed at .40% agar concentration (Weiss motility assay 3/11/07) -LWeiss

+

# Add 25mL purified water to the flask and swirl to dissolve

+

# Autoclave 25 minutes with slow exhaust

+

# Upon cooling, add appropriate antibiotics and inducers

+

*Kan 50µg/mL

+

*Amp 100µg/mL

+

*HSL between 10-4 - 10-8

+

*IPTG 1mM

+

# Allow plates to solidify for ~3 hours before use

+

*Plates can be prepared the previous day before inoculated, but tend to try out if prepared too far in advance-LW

+

+

Day 1: Inoculate O'N Culture

+

# Inoculate 3mL overnight cultures of all samples to be tested

+

# Shake at 200 RPM in 30°C incubator

+

*I have done this at 37°C and it seems to be ok, I would recommend shaking at whatever temperature the motility assay will be run at-LW

+

+

Day 2: Assay

+

# Dilute overnight culture 1:100 in LB (30uL of culture in 3mL LB)

+

# Grow in a 30°C incubator at 200 RPM to an A590nm O.D. between 0.3-0.4

.40% Eiken Agar with sender produced AHL tagged for degradation after 8 hours

.40% Eiken Agar with regular sender and receiver cells after 8 hours

Observations:

.20% agar plate difficult to handle as agar was extremely fragile. Results were diminished by heavy diffusion.
.30% agar results in fair motility and is much easier to handle
.40% agar results in no motility

April 28, 2007: Synthetic Biology 3.0

May 15, 2007: General Procedures

These are all the general cloning kinds of things that will be used to build our constructs. We will probably get into getting parts sequenced if things do not seem to be working, but that gets expensive and takes a couple of days, so my thoughts are we should save that until it looks like we are getting into trouble. Labeling is key.

Labeling

First note this is the most important thing for how easy it is. In the past we have had a lot of problems however so I have come up with a system. Remember everyone is going to need to be able to read this, so use a fine tipped sharpie always. Also EtOH is an organic solvent and will remove your title. Be careful with this especially during minipreps. Write on the outside of the tube, Thats really obvious it would seem, but just a warning.

Microcentrifuge tubes
On Cap:

Row 1: Initials Date

Row 2: Part/parts (this is a quick reference, always know what it is that you are using)

Row 3: ID number 0001-9999 (each member will have a list of every tube they have online)

Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge

allign tubes such that the cap clip is facing outward

decant supernatant into spin column

Centrifuge for 60s. Discard flow-through.

Add .75mL Buffer PE and centifuge for 60s.

Discard flowthrough and centrifuge for 60s

Place the spin column into a new 1.5mL microcentrifuge tube.

Add 30-50uL of water (8.5>pH>7) to the center of each spin column.

Let tubes stand vertically for 1min, then centrifuge for 1min.

Digest

Add components generally by largest volume to smallest. I tend to add water first just so I do not waste pipettips if I am using different vectors, but cetainly do not start with .5 or 1 uL components.

Choosing your Enzymes: The biobricks are built with the restriction sites as following: -EcoR1-Xba1-Part-Spe1-Pst1- it is important to remember that your plasmid may have some of these restriction sites too, so if too many bands are appearing on the gel becareful of this. Keep these enzymes on ice when they are out of the feezer, and put them back immediately after you use them. If enzymes happen to be left out please let me know and we will decide what to do with them. Do not put enzymes that have been left out back in the freezer, or future restrictions will not work.

Choosing your buffer: Check the compatibility with each enzyme and the buffer. There should be a list for double digestions.

Forgetting to add BSA will make the whole thing not work.

Examples demonstrate the two main digets that will be used in this lab.

EX1-10uL Digest

3.0uL H2O

4.0uL Vector

1.0uL Buffer X

1.0uL BSA

0.5uL Enzyme 1

0.5uL Enzyme 2

EX2-20uL Digest

4.0uL H2O

11.uL Vector

2.0uL Buffer X

2.0uL BSA

0.5uL Enzyme 1

0.5uL Enzyme 2

Running a Gel
TAE Buffer-if you are extracting from your gel
TBE Buffer-for better imaging
Do not load DNA in high concentrations. It may be tempting to put in as much concentrated DNA as you can, but this is not a good thing.

Gel Extraction

Ligation

Transformation

May 21, 2007: Summer Schedule and Restriction Enzymes

Summer Schedule and Week's Plan

Slide1

Slide2

Slide3

Slide4

Slide5

Slide6

Slide7

Slide8

Slide9

Restriction Enzymes

|---------EcoRI--------|

|---------XbaI---------|

|---------PART---------|

|---------SpeI---------|

|---------PstI---------|

5' G|AATT C 3'

5' T|CTAG A 3'

5' A|CTAG T 3'

5' C TGCA|G 3'

3' C TTAA|G 5'

3' A GATC|T 5'

3' T GATC|A 5'

3' G|ACGT C 5'

May 22, 2007: Growing Parts From Registry Plates

Registry plates are kept in the -4 degree freezer in a box labeled registry plates.

Take a 10uL pipetment and pipet 10uL of dd-water up and down inside target well on the registry plate

Once the water appears red place that in a labeled microcentrifuge tube (Initials Date, Part name well #, and ID number).

Now the registry part is ready to be transformed. Proceed with regular transformation.

July 17, 2007: Giant Digest

July 23, 2007: Motility Assay Protocol

Motility Protocol:

For each plate to be made, add the following to a 125mL flask:

0.25g Bacto tryptone

0.20g NaCl

0.15g sodium citrate

0.075g Eiken agar (for a 0.30% agar swarm plate)

note: <.25% plates are difficult to deal with as they break very easily (Weiss motility assay 3/11/07) .26%-.32% is what I tend to use. Cells did not swarmed at .40% agar concentration (Weiss motility assay 3/11/07) -LWeiss

Add 25mL purified water to the flask and swirl to dissolve

Autoclave 25 minutes with slow exhaust

Upon cooling, add appropriate antibiotics and inducers

Kan 50µg/mL

Amp 100µg/mL

HSL between 10-4 - 10-8

IPTG 1mM

Allow plates to solidify for ~3 hours before use

Plates can be prepared the previous day before inoculated, but tend to try out if prepared too far in advance-LW

Day 1: Inoculate O'N Culture

Inoculate 3mL overnight cultures of all samples to be tested

Shake at 200 RPM in 30°C incubator

I have done this at 37°C and it seems to be ok, I would recommend shaking at whatever temperature the motility assay will be run at-LW